Creba isoform

ABSTRACT

The present invention relates generally to a novel CREBa polypeptide isoform, polynucleotides encoding the polypeptide, expression constructs comprising the polynucleotides, host cell transformed or transfected with the polynucleotides, methods for producing the polypeptide, and methods to identify inhibitors of binding between the CREBa and other polypeptides or polynucleotides.

This application is a continuation of U.S. patent application Ser. No.:09/005,970, filed Jan. 12, 1998 which issued as U.S. Pat. No.:5,959,079, which is a divisional of U.S. patent application Ser. No.:08/721,684 filed Sep. 27, 1996, which issued as U.S. Pat. No.:5,854,016.

FIELD OF THE INVENTION

The present invention relates to novel polynucleotides encodingpolypeptides which bind to cAMP regulatory DNA sequences.

BACKGROUND OF THE INVENTION

Extracellular signal transduction leading to specific gene expression isoften carried out by a series of enzymatic reactions which ultimatelymodulate activity of nuclear transcription factors. In one such example,extracellular signaling alters cytoplasmic levels of adenosine 3′,5′-monophosphate (cAP) which in turn modulates levels of activecAMP-dependent protein kinase (PKA). Once activated, PKA migrates intothe nucleus and phosphorylates transcription factors which recognize DNAsequences common to genes that are regulated by cAMP signaling pathways.The common DNA sequence which permits cAMP regulation of gene expressionhas been designated the cAMP regulatory element (CRE) and thetranscription factors which recognize and bind to the CRE are known asCRE-binding (CREB) proteins. It has been proposed that CREB proteins areordinarily found in association with CRE DNA sequences and that thephosphorylation state of CREB determines the degree to which the proteinis capable of inducing transcription of the associated gene. Oncephosphorylated, CREB is able to bind a CREB-binding protein (CBP) whichpermits interaction of the complex with transcription factor TFIIB.

It is therefore apparent that regulation of the phosphorylation state ofCREB is central to specific gene expression by cAM. The phosphorylationstate of CREB, however, is not regulated solely by PKA. On the contrary,the degree of CREB phosphorylation is balanced between the activities ofphophatases as well as kinases other than PKA. Thus, while CREB is amajor participant in coordination of cAMP gene expression, CREB activityis subject to concurrent control by enzymes in other, non-cAMP relatedpathways.

Members in the CREB family of proteins contain conserved regions whichcarry out specific functions related to transcriptional activation. Atthe carboxy terminus, all CREB proteins have a leucine zipper regionwhich permits dimerization of CREB with other CREB proteins or otherheterologous transcription factor subunits. Adjacent the leucine zipperregion, CREB proteins are characterized by a region designated thekinase inducible domain (KID) which is subject to phosphorylation bymultiple kinases other than PKA, including for example, protein kinase C(PKC), casein kinase I (CKI) and casein kinase II (CKII), and possiblycalcium-calmodulin dependent kinases I and II. At the amino terminus,CREB proteins each contain a DNA binding domain rich in basic aminoacids. Despite seemingly subtle differences between proteins within thefamily, reports of variation in gene expression suggest that theproteins have unique physiological roles.

BRIEF SUMMARY OF THE INVENTION

In one respect the present invention provides purified and isolatedpolynucleotides (e.g., DNA sequences, RNA transcripts thereof andanti-sense oligonucleotides thereof) encoding a novel mouse cAMPregulatory element binding, designated mCREIBa, polypeptide well aspolypeptide variants (including fragments and deletion, substitution,and addition analogs) thereof which display one or more DNA or proteinbinding activities, one or more specific gene transcription modulationactivities, and/or immunological properties specific to mCREBa. DNAbinding properties include recognition of specific DNA sequences throughwhich mCREBa is able to modulate specific gene expression, while proteinbinding properties include interaction with various regulators of mCREBaactivity, including any of a number of protein kinases, as well asinteractions with specific and non-specific transcription factors.Preferred DNA sequences of the invention include genomic and cDNAsequences as well as wholly or partially chemically synthesized DNAsequences. A presently preferred olynucleotide is set out in SEQ IDNO: 1. Plasmid pBSmb3, comprising the preferred cDNA of the invention,in E. coli strain DH5αF′ was deposited on September 18, 1996 with theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, and assigned Accession Number 98171. Biological replicas (i.e.,copies of isolated DNA sequences made in vivo or in vitro) of DNAsequences of the invention are contemplated. Also provided areautonomously replicating recombinant expression constructs such asplasmid and viral DNA vectors incorporating mCREBa sequences andespecially vectors wherein DNA encoding mCREBa or a mCREBa variant isoperatively linked to an endogenous or exogenous expression control DNAsequence.

According to another aspect of the invention, host cells, especiallyunicellular host cells such as procaryotic and eucaryotic cells, arestably transformed with DNA sequences of the invention in a mannerallowing the desired polypeptides to be expressed therein. Host cells ofthe invention are conspicuously useful in methods for the large scaleproduction of mCREBa and mCREBa variants wherein the cells are grown ina suitable culture medium and the desired polypeptide products areisolated from the cells or from the medium in which the cells are grown.

Novel mCREBa polypeptides of the invention may be obtained as isolatesfrom natural cell sources, but, along with mCREBa variant products, arepreferably produced by recombinant procedures involving host cells ofthe invention. A presently preferred amino acid sequence for a mCREBapolypeptide is set out in SEQ ID NO: 2. The recombinant products may beobtained in fully or partially phosphorylated or dephosphorylated forms,depending on the cell selected for recombinant expression and/orpost-isolation processing. The mCREBa variants of the invention maycomprise mCREBa fragments which include all or part of one or more ofthe domain regions having a biological or immunological property ofmCREBa including, e.g., the ability to bind to a polypeptide orpolynucleotide binding partner of mCREBa and/or inhibit binding ofmCREBa to a natural binding partner. The mCREBa variants of theinvention may also comprise polypeptide analogs wherein one or more ofthe specified amino acids is deleted or replaced: (1) without loss, andpreferably with enhancement, of one or more biological activities orimmunological characteristics specific for mCREBa; or (2) with specificdisablement of a particular polypeptide/polypeptide orpolypeptide/polynucleotide binding function. Analog polypeptidesincluding additional amino acid (e.g., lysine or cysteine) residues thatfacilitate multimer formation are contemplated. Variant mCREBapolypeptides further include fusion polypeptides wherein all or part ofmCREBa is expressed in conjunction with extraneous polypeptidesequences, including, but not limited to, for example poly-histidinetags, biotinylation tags, β-galactosidase chimera, or chimericpolypeptides including one or more the DNA binding or transactivatingdomains from various transcription factors.

Also comprehended by the present invention are antibody substances(e.g., monoclonal and polyclonal antibodies, antibody fragments, singlechain antibodies, chimeric antibodies, CDR-grafted antibodies and thelike) and other binding proteins (e.g., polypeptides and peptides) whichare specific (i.e., non-reactive with previously identified CREBaisoforms to which mCREBa is structurally related) for mCREBa or mCREBavariants. Antibody substances can be developed using isolated natural orrecombinant mCREBa or mCREBa variants. Binding proteins of the inventionare additionally useful for characterization of DNA or polypeptidebinding site structure(s) (e.g., epitopes and/or sensitivity of bindingproperties to modifications in mCREBa amino acid sequence).

Binding proteins are useful, in turn, in compositions for immunizationas well as for purifying polypeptides of the invention and identifyingcells which express the polypeptides on their surfaces. They are alsomanifestly useful in modulating (i.e., blocking, inhibiting orstimulating) DNA and/or polypeptide binding biological activitiesinvolving mCREBa, especially those mCREBa effector functions involved inspecific and non-specific gene expression. Anti-idiotypic antibodiesspecific for anti-mCREBa antibody substances and uses of suchanti-idiotypic antibody substances in modulating gene expression arealso contemplated. Assays for the detection and quantification of mCREBain body fluids, such as serum or cerebrospinal fluid, may involve, forexample, a single antibody substance or multiple antibody substances ina “sandwich” assay format.

The scientific value of the information contributed through thedisclosures of DNA and amino acid sequences of the present invention ismanifest. As one series of examples, knowledge of the sequence of a cDNAfor mCREBa makes possible the isolation by DNA/DNA hybridization ofgenomic DNA sequences encoding mCREBa and specifying mCREBa expressioncontrol regulatory sequences such as promoters, operators and the like.DNA/DNA hybridization procedures carried out with DNA sequences of theinvention and under stringent conditions are likewise expected to allowthe isolation of DNAs encoding allelic variants of mCREBa, otherstructurally related proteins sharing one or more of the biologicaland/or immunological properties specific to mCREBa, and proteinshomologous to mCREBa from other species. DNAs of the invention areuseful in DNA/RNA hybridization assays to detect the capacity of cellsto express mCREBa. Also made available by the invention are anti-sensepolynucleotides relevant to regulating expression of mCREBa by thosecells which ordinarily express the same. As another series of examples,knowledge of the DNA and amino acid sequences of mCREBa makes possiblethe generation by recombinant means of mCREBa variants such as hybridfusion proteins characterized by the presence of mCREBa proteinsequences in association with various extraneous polypeptide sequences.

The DNA of the invention also permits identification of novel genesencoding binding partner polypeptides for mCREBa in any of a number ofwell known techniques, including for example, di-hybrid screening andgel overlay assays. The DNA and amino acid sequence information providedby the present invention also makes possible the systematic analysis ofthe structure and function of mCREBa and identification of thosemolecules with which it will interact. The idiotypes of anti-mCREBamonoclonal antibodies of the invention are representative of suchmolecules and may mimic natural binding proteins (peptides andpolypeptides) through which mCREBa biological activities are modulatedor by which mCREBa modulates intracellular events. Alternately, they mayrepresent new classes of modulators of mCREBa activities. Anti-idiotypicantibodies, in turn, may represent new classes of biologically activemCREBa equivalents. In vitro assays for identifying antibodies or othercompounds that modulate the activity of mCREBa may involve, for example,immobilizing mCREBa or a natural binding polypeptide or polynucleotideto which mCREBa binds, detectably labelling the nonimmobilized bindingpartner, incubating the binding partners together and determining theeffect of a test compound on the amount of label bound wherein areduction in the label bound in the presence of the test compoundcompared to the amount of label bound in the absence of the testcompound indicates that the test agent is an inhibitor of mCREBabinding.

In the alternative, DNA of the invention permits identification ofinhibitors of mCREBa binding to other natural binding partners thoughutilization of a split hybrid assay, wherein mCREBa, or a proteinbinding domain fragment thereof, is expressed in a host cell as a fusionprotein in combination with either a DNA binding or transactivatingdomain of one or more transcription factors. A natural binding partnerof mCREBa is also expressed in the same host cell as a fusion protein incombination with either a DNA binding or transactivating domain of atranscription factor (whichever is not incorporated into the mCREBafusion protein). Expression of the two fusion proteins, and subsequentassociation of the binding partners permits association of the DNAbinding and transactivating domains forming an active transcriptionfactor that leads to expression of a repressor protein. The expressedrepressor protein, in turn, prevents expression of a reporter gene, thussignificantly decreasing survival of the host cell. The thus transformedhost cells can then be contacted with putative inhibitors ofmCREBa/binding partner interaction and actual inhibitors identified asthose which prevent mCREBa binding to its partner protein, thuspreventing expression of the repressor protein which cannot thereforeblock expression of the reporter gene. An inhibitor therefore indirectlyprovides for a positive signal generated by expression and detection ofthe particular reporter gene product. There are at least three differenttypes of libraries used for the identification of small moleculemodulators. These include: (1) chemical libraries, (2) natural productlibraries, and (3) combinatorial libraries comprised of random peptides,oligonucleotides or organic molecules. Chemical libraries consist ofstructural analogs of known compounds or compounds that are identifiedas inhibitory via natural product screening. Natural product librariesare collections of microorganisms, animals, plants, or marine organismswhich are used to create mixtures for screening by: (1) fermentation andextraction of broths from soil, plant or marine microorganisms or (2)extraction of plants or marine organisms. Combinatorial libraries arecomposed of large numbers of peptides, oligonucleotides, or organiccompounds as a mixture. They are relatively easy to prepare bytraditional automated synthesis methods, PCR cloning, or proprietarysynthetic methods. Of particular interest are peptide andoligonucleotide combinatorial libraries. Still other libraries ofinterest include peptide, protein, peptidomimetic, multiparallelsynthetic collection, recombinatorial, polypeptide libraries.

The DNA sequence information provided by the present invention alsomakes possible the development, by homologous recombination or“knockout” strategies [see, e.g., Kapecchi, Science, 244: 1288-1292(1989)], of rodents or rabbits that fail to express a functional mCREBaprotein or that express a variant mCREBa protein. Such rodents areuseful as models for studying the activities of mCREBa and mCREBamodulators in vivo.

DETAILED SUMMARY OF THE INVENTION

The present invention is illustrated by way of the following examples.Example 1 describes isolation of a partial cDNA encoding a novel proteinby virtue of its interaction with CK1b. Example 2 provides furthercharacterization of the interaction between the novel protein and CKIδ.Example 3 relates to examination of tissue distribution of the novelmCREBa polypeptide. Example 4 addresses isolation of a full length cDNAencoding mCREBa. Example 5 describes generation of antibodiesimmunospecific for mCREBa. Example 6 relates to recombinant expressionof mCREBa. Example 7 describes identification of the DNA binding sitefor mCREBa. Example 8 relates to generation of a high throughputscreening assay for identification of modulators of mCREBa/DNA bindingactivity. Example 9 addresses use of mCREBa in a split hybrid assay toidentify inhibitors of mCREBa/protein interactions.

EXAMPLE 1 Isolation of cDNA Encoding A Protein That Interacts with CKIδ

In order to isolate cDNAs encoding proteins that interact with CKIδ, atwo-hybrid screen was employed using an expression vector encoding aLexA-CKIδ fusion protein as bait. DNA encoding CKIδ was subcloned intothe BamHI site of pBTM116 [Bartel, et al., in Cellular Interactions inDevelopment: a Practical Approach, Hartley (ed.), IRL Press; Oxford, pp.153-179 (1993)] by the following method. The coding region of CKIδHU wasinitially modified to incorporate BamHI restriction sites using PCR withprimers EC 140 (SEQ ID NO: 3) and ECI41 (SEQ ID NO: 4) which produced a1.3 kbp DNA fragment.

CGCGGATCCTAATGGAGCTGAGAGTCGGG SEQ ID NO: 3

CGCGGATCCGCTCATCGGTGCACGACAGA SEQ ID NO: 4

The amplification reaction included 300 ng CKIδHU template DNA, 1X PCRbuffer (Perkin Elmer-Cetus), 1.5 mM MgCl₂, 200 uM each dATP, dCTP, dGTP,and dTTP, 10 ng/ml each primer ande 1 unit AmpliTaq (PerkinElmer-Cetus). The reaction was carried out with an initial incubation at94° C. for four minutes, followed by thirty cycles of incubation at 94°C for one minute, 50° C. for two minutes, and 72° C. for four minutes.The resulting PCR product was digested with BamHI and ligated intoplasmid pAS1 [Durfee, el al., Genes and Development 7:555-569 (1993)]such that the encoded protein would be expressed containing an influenzahemagglutinin (HA) epitope tag. The resulting plasmid was transformedinto E. coil by standard procedures and expression of the CKIδ fusionprotein was confirmed by immunoblot using monoclonal antibody 12CA7(Boehringer Mannheim) immunospecific for the HA epitope. The BamHIfragment encoding CKIδ was subsequently subcloned into the BamHI site ofpBTM 116 to give plasmid pBTMCKIδ which was transformed into an S.cerevisiae strain L40 (MATa his3 Δ200 trp1-901 leu2-3,112 ade2 LYS2::(OexAop)₄ H1S3 L TRA3::(lexAop)₈,-lacZ GAL4) to generate strain CKIδ/L40.CKIδ40 was subjected to a large scale transformation with a cDNA librarymade from mouse embryos staged at days 9.5 and 10.5. The cDNA librarywas prepared in vector pVP16 according to the method of Hollenberg, etal. [Mol. Cell. Biol. 15:3813-3822 (1995)]. Approximately 40 milliontransformants were obtained as determined by survival on media lackingleucine and tryptophan.

Eighty-eight million transformants were assayed for protein/proteininteraction by plating on selective media lacking leucine, tryptophan,and histidine. The ability of the yeast to grow in the absence ofhistidine suggested an interaction between CKIδ and a protein encoded bya library sequence. Colonies capable of growth on media lackinghistidine were further screened by standard methods for the ability toexpress β-galactosidase encoded by a gene under transcriptional controlof the LexA operator. One hundred of the yeast colonies that turned bluemost rapidly were grown in liquid media lacking leucine and tryptophanand total yeast DNA was prepared by standard methods. The total yeastDNA was used to transform the E. coli strain C600, which lacks theability to grow on media lacking leucine unless a plasmid expressingleucine is prese - ft. Bacteria were plated on agar containingcarbenicillin (an ampicillin derivative) and lacking leucine. Individualcolonies that grew under these conditions were grown up and plasmid DNAprepared.

Since many false positives can occur, positive plasmids were retestedfor interaction with the LexA-CKIδ fusion protein, as well as with aLexA-lamin fusion protein as a negative control. Briefly, the isolatedcDNA library plasmid was cotransformed into L40 with either a plasmidencoding LexA-CKIδ or LexA-lamin, three yeast colonies from eachtransformation are picked and tested in a standard β-galactosidaseblue/white assay. Cells that turned blue with LexA-CKIδ but not withLexA-lamin were grown up, the plasmid DNA isolated, and the sequencedetermined by standard methods. Search of the National Center forBiotechnology Information data base indicated three classes of cDNAswere obtained. Collectively, proteins encoded by the DNAs weredesignated CKIδ-delta-interacting proteins or DIPS.

One class of cDNAs, typified by a clone designated DIP25, contained aportion of a cDNA that was related, but not identical to Drosophilatranscription factor dCREBa. The fill length clone was thereforedesignated mCREBa and contains two distinct amino acid motifscharacteristic of CREB proteins. A region rich in basic amino acids,presumably a DNA binding region, is located at the amino terminus whilea leucine zipper domain is found at the carboxyl terminus. A secondclass of DNAs was nearly identical to an EST clone T81950, while thethird class showed no homology to any sequences in the databases.

Example 2 Characterization Of The Interaction Between the mCREBa DIP25Clone and CKIδ

In order to further characterize the interaction between CKIδ and themCREBa DIP25 fragment, the mouse clone was expressed in E. coli as a GSTfusion protein as described below.

DIP25-encoding DNA was digested with BamHI and EcoRI and anapproximately 500 bp fragment was isolated and ligated into plasmidpGEX3T (Pharmacia, city state) previously digested with BamHI and EcoRI.The resulting plasmid was transformed into E. coli. Protein expressionwas induced with addition of 0.2 mM IPTG to the media and a GST-DIP25protein of approximately 45 kDa was expressed. The protein was purifiedby adsorption on glutathione agarose (Pharmacia) and used to assess thebinding of DIP25 to CK1 6 in vitro as follows.

Recombinant CKIδ was produced as a histidine-tagged fusion protein inE.coli,[Ausubel, et al., Current Protocols in Molecular Biology, JohnWiley & Sons (1993) pp. 10.11.8-10.11.22]. Briefly, CKIδ was cloned intothe expression vector pET15b (Novagen). An NdeI restriction site wascreated at the 5′end of CKIδ by site directed mutagenesis and anNdeI/BamHI CKIδ fragment was ligated into pET15b previously digestedwith the same two enzymes. Site directed mutagenesis was carried outwith an oligonucleotide designated KME18 (SEQ ID NO: 10) using theMutagene Phagemid In Vitro Mutagenesis Kit, Version 2 (BioRad) accordingto manufacturer's suggested protocol.

GAATCGGGCCGCCGAGATCTCATATGGAGCTGAGAGTC   SEQ ID NO: 10

The resulting plasmid was transformed into bacterial strain BL21DE3 andthe cells grown to optical density (A₆₀₀) of 0.6. The cells were inducedwith 1 MM IPTG for 18 hours at 25° C., harvested, resuspended in breakbuffer (containing 20 mM NaPO₄, pH 8, 300 mM NaCI, 1 mM PMSF, 1 μMaprotein, 1 μM leupeptin) and lysed in a French Press. The lysate wascentrifuged at 100,000 X g for 60 minutes and the supernatant loadedonto a Ni-NTA-Agarose column (QIAGEN, Germany). The column was washedwith break buffer and protein eluted using a gradient of 0 to 200 mMimidazole. CKI activity associated with CKIδ eluted between 50 and 80 mMimidazole. The histidine-tagged fusion protein was incubated at 4° C. inbinding buffer (50 mM NaCI, 10 mM Tris pH 7.5, 10% glycerol, 0.1%Triton) with the DIP25-GST fusion protein immobilized on glutathioneagarose beads. After an hour incubation, the beads were washed threetimes in binding buffer and bound protein eluted by boiling in SDSsample buffer containing 2% SDS, 20 mM Tris, pH 6.8, 20% glycerol, and0.001% bromphenol blue. A minor fraction of the DIP25-GST fusion proteinbound the histidine- tagged CKIδ while no CKIδ bound to the negativecontrol, immobilized GST alone. The inefficient binding may reflecteither improper folding of the GST fusion protein or it may indicate aneed for some post-translational modification of DIP25 that E. coli areunable to carry out.

Example 3 Tissue Distribution of mCREBa

In order to determine the tissue expression pattern of mCREBa mRNA,Northern blot analysis was carried out using containing a commercialmembrane containing poly A⁺RNA isolated from various mouse tissues andfrom whole mouse embryos at days 7, 11, 15, and 17 in gestation(Clontech, Palo Alto, Calif.). The membrane was probed following themanufacturer's suggested protocol using DNA generated by PCR from themCREBa clone using the primers mCR-1 (SEQ ID NO: 5) and mCR-2 (SEQ IDNO: 6).

GGAATTCGCTCAAGGAGAGTCCTATTGG   (SEQ ID NO: 6)

CGGGATCCTCACAGCTCCACATAAGCTGC   (SEQ ID NO: 5)

The PCR included 50 ng DIP25 DNA as template, 1 X PCR buffer(Perkin-Elmer Cetus), 1.5 MM MgCI₂, 200 μM dATP, 200 μM dGTP, 200 μMdTTP, 1 μM dCTP, 50 μCi α³²P-dCTP, 10 ng/ml each primer, and 1 unitAmpliTaq (Perkin-Elmer Cetus). The reaction was carried out in aPerkin-Elmer Cetus Thermocycler Model 480 as follows: an initialdenaturation cycle at 94° C. for four minutes, followed by 20 cycles of94° C for 15 seconds, 60° C. for 15 seconds, and 72° C. for 30 seconds.Unincorporated nucleotides were removed using a Nuc-trap Push Column(Stratagene).

Two mRNAs were detected with one migrating at about 8 kb and the otherat about 3.75 kb. The two mRNAs always detected together suggesting twosplice variants. On the tissue RNA membrane, the highest levels ofexpression were seen in kidney, lung, and heart, with lower levelsdetected in skeletal muscle, liver, brain and testis. Very lowexpression was detected in spleen RNA on this particular blot. In themouse embryo samples, expression of both the 8 kb and the 3.75 kb mnRNAswas highest at day 7 and lowest at day 11. After day 11, a progressiveincrease in expression was detected up to days 15 and 17.

In situ hybridization of mouse embryos was performed to determine whichembryonic tissues expressed the mCREBa mRNA. Normal Balb/c mouse embryoswere harvested and embedded in optimal temperature compound (TissueTech, Elkhart, Ind.) and whole embryos were sectioned at 6 micronthickness. Tissue sections were placed on Superfrost Plus® (VWRScientific, Seattle, Wash.) and allowed to air dry overnight at roomtemperature. Sections not used immediately thereafter were stored at−70° C. After drying, sections were fixed in 4% paraformaldehyde (Sigma)in PBS for 20 minutes at room temperature, dehydrated using ethanol inincreasing concentrations (70%, 95% and finally 100%) for one minute at4° C. at each concentration, and air dried at room temperature. Thesections were denatured for two minutes at 70° C. in a solutioncontaining 70% formamide in 2X SSC. Sections were rinsed in 2X SSC at 4°C. and dehydrated and air dried again as previously described.Hybridization was carried out using ³⁵S-labeled single stranded mRNAgenerated from murine DIP25 DNA by in vitro transcription using ³⁵S-dUTP(Amersham). The labeled probe and diethylpyrocarbonate (DEPC)-treatedwater were added to a hybridization buffer of 50% formamide, 0.3 M NaCl,20 mM Tris, pH 7.5, 10% dextran sulfate, 1 X Denhardt's, 100 mMdithiothreitol (DTT) and 5 mM EDTA. Prior to addition, the probesolution was heated for three minutes at 95° C. to denature the probe,and 20 μl of the buffer was applied to each tissue section which wasthen covered with a sterile RNAse free coverslip. Hybridization wascarried out overnight at 50° C.

After hybridization, the sections were washed for one hour at roomtemperature in 4X SSC with 10 mM DTT, followed by additional washes for40 minutes at 60° C. in a buffer containing 50% deionized formamide, 2XSSC, and 10 mM DTT and 30 minutes at room temperature in a 0.1 X SSCbuffer. The sections were dehydrated and air dried as described aboveand dipped in Kodak (Rochester, N.Y.) NTB2 nuclear emulsion diluted 1:1with 0.6 M ammonium acetate at 45° C. Slides were air dried 1 to 2 hoursin the dark and exposed at 4° C. in the dark in the presence of adesiccant. After ten days exposure, the slides were developed in KodakDektol developer, washed with deionized water and submerged in Kodakfixer for four minutes at room temperature. The sections were thencounterstained with hematoxylin and eosin (Sigma, St. Louis, Mo.).Results from this analysis are shown in Table 1.

TABLE 1 Expression of mCREBa mRNA in mouse embryos Day 15 Day 16 Day 17Day 18 spinal roots + + + + peripheral nerves + + + + hindbrain + nasalareas + + optic areas + + + unidentified region + + + of the braintongue + bladder + + kidney + lung +

Example 4 Isolation of a cDNA Encoding Full Length mCREBa

In order to obtain a full length cDNA encoding mCREBa, 5.4×10⁶ clonesfrom a mouse brain UniZAP XR cDNA library (Stratagene) were screened byhybridization with a probe generated by PCR from the mCREBa DIP25 cloneusing oligos mCR-1 and mCR-2 as described above. Hybridization wascarried under conditions wherein nitrocellulose filters were incubatedfor 18 hours at 65° C. in 3X SSC, 5X Denhardt's, 0.1% sarkosyl, 20 mMNaPO₄, pH 6.8, 100 μg/ml single stranded salmon sperm DNA. One clonehybridized to the probe. The cDNA, designated pBSmb3, was isolated by invivo excision according to the manufacturers suggested protocol andsubjected to microsequencing. Sequencing analysis of the cDNA revealed a3190 bp clone containing an open reading frame beginning at nucleotide304 and ending at nucleotide 1866 with a predicted molecular weight ofapproximately 57 kDa. The nucleotide and amino acid sequences of theclone are set forth in SEQ ID NOs: 1 and 2, respectively. The DIP25clone corresponds to nucleotides 1010 through 1514 of the full lengthmCREBa clone pBSmb3, encoding amino acids 237 through 405 representingboth the basic DNA binding domain and the leucine zipper domain of theprotein. Comparison of amino acids 406-508 of the leucine zipper regionin dCREBa to amino acids 260 to 361 in the corresponding region ofmCREBa reveals 78% identity and comparison of these regions atnucleotide level indicates 68% homology between nucleotides 2202 to 2511of dCREBa and nucleotides 1081 to 1386 of mCREBa. In vitro transcriptionand translation of the clone using TNT kit (Promega, Madison, Wis.)produced a protein product that migrated slightly slower than thepredicted molecular weight (about 75 kDa), possibly caused by the highlycharged basic DNA binding domain or the secondary structure formed inthe leucine zipper domain. In addition, a sub clone of pBSmb3 wasgenerated by digesting the plasmid with EcoRI and XhoI to produce a 1.8kb fragment that was ligateded into pBluescript sk⁻ previously digestedwith the same two enzymes. The resulting plasmid was designatedpBSmb3E/X. In vitro transcription and translation of the encoded generesulting in production of a polypeptide of approximately 75 kDsuggesting that the entire coding sequences for the protein wascontained on the 1.8 kb fragment. Further, amino acid sequence analysissuggests that, similar to other CREB isoforms, mCREBa may potentially bephosphorylated by kinases other than CKIδ, including but not limited toother CKI isoforms, CKII, cdc2, MAP kinase, and S6 kinase.

Example 5 Production and Characterization of mCREBa Antibodies

Polyclonal antibodies specific for mCREBa were generated using theDIP25-GST expression product of plasmid pGEX3T-DIP25 described above.Briefly, C600 bacteria transformed with the plasmid were grown to anabsorbance at 600 nm of 1.8, at which point expression of DIP25-GST wasinduced for five hours by addition of 0.1 mM IPTG which acted on theendogenous lac promoter. Following induction, bacteria were harvested bycentrifugation and resuspended in phosphate buffered saline (PBS)containing 1 mM pbenylmethylsulfonylfluoride (PMSF), 1μg/ml leupeptide,and 1 μg/ml pepstatin. The cells were lysed in a French press andcentrifuged for twenty minutes at 12,000 x g. The supernatant wasincubated for one hour with 3 ml of a 50% slurry ofglutathione-Sepharose 4B (Pharmacia), after which the resin was pelletedand washed three times in PBS. Protein was eluted from the resin using50 mM glutathione in 10 mM Tris, pH 8.0.

Female New Zealand White rabbits were initially immunized with 200 μgDIP25-GST antigen mixed with Freund's complete adjuvant injectedsubcutaneously at multiple sites. Subsequent boosters were carried outwith protein that was first boiled in SDS sample buffer, loaded onto anSDS polyacrylamide gel, and electroeluted from gel slices. Boosterantigen preparation was mixed with Fruend's incomplete adjuvant andadministered at approximately 21 day intervals following the firstimmunization. Test bleeds taken after immunizations 3, 4, and 5.

The immunization resulted in two polyclonal antisera designated 6179 and6144. In order to determine if the antisera recognized mCREBa, pBSmb3was transcribed and translated in vitro in the presence of³⁵S-methionine using a TNT kit (Promega). The translation extract wasdiluted to 0.5 ml with NP40 IBP (containing 1% Nonidet P40, 50 mM Tris,pH 7.5, 100 mM NaCI, 1 mM EDTA) and incubated on ice for one hour with 5μl rabbit antisera. Following incubation, 20 μl of a 50% slurry ofprotein A agarose (Repligen) was added and incubation continued on icefor an additional 20 minutes. Immune complexes were collected bycentrifugation and washed three times in NP40 IPB. Protein was elutedfrom the complexes by boiling in SDS sample buffer and loaded onto anSDS polyacrylamide gel. After separation, the gel was fixed in aceticacid and methanol, treated with the fluor Amplify (Amersham), dried, andexposed to x-ray film. Autoradiography indicated that both antiserareacted with the 79 kDa expression product from pBSmb3E/X describedabove.

In order to determine whether the antisera reacted with mCREB expressedin mammalian cells, an expression plasnid encoding a mammalian CREB wasconstructed as follows. Parental plasmid pcDNA 3 (Invitrogen) wasdigested with restriction enzymes EcoRI and XhoI and ligated with the1.8 kbp EcoRI/XhoI fragment from pBS rmb3E/X described above andencoding the complete mCREB protein. The resulting plasmid was used totransiently transform a human embryonic kidney cell line 293T by themethod of Chen and Okayama [Biotechniques 6:632-638 (1988)]. Celllysates were generated 48 hours after transformation in buffercontaining 1% Trito ,. X-100, 10 MM Tris, pH 7.6,5 mM EDTA, 50 mM NaCI,30 mM Na₄P₂O₇, 50 mM NaF, 100 μM Na₃VO₄, 1 mM PMSF, 1 μg/ml aprotein,and 1 μg/ml leupeptin and the lysate centrifuged at 10,000 x g. Onehundred pg cleared lysate was loaded onto a 10% SDS polyacrylamide geland immunoblots were produced by standard techniques. Both antisera,diluted 1:1000, reacted with a protein of approximately 79 kDa proteinin the in lysate from cells transfected with pcDNA3 containing the mCREBsequences.

Example 6 Expression of Recombinant mCREBa

Recombinant mCREBa was expressed in E. coli as a fusion with a proteinusing a modified Pinpoint expression vector (Promega) which permitsexpression of a biotinylated protein when the host cells are grown inthe presence of biotin. Briefly, a 1.8 kb NcoI/XhoI fragment was excisedfrom pBSmb3 and subcloned into expression plasmid araBC previouslydigested with NcoI and XhoI. The resulting intermediate plasmidcontained mCREBa-encoding sequences under the control of the arabinosepromoter. In order to fuse a biotin tag to mCREB, an EcoRI/NcoI promoterfragment from the expression plasmid arabio1b was inserted into theintermediate plasmid such that the mCREBa coding sequence was in framewith a biotin tag and under the control of the arabinose promoter. Theresulting plasmid was designated arabiomCREB. Bacteria were transformedwith arabiomCREB by standard techniques and grown to mid-log phase inLuria Broth containing 2 μM biotin. Expression of biotin-mCREBa wasinduced with 1% arabinose, the bacteria harvested by centrifugation, andthe pellet was resuspended in 50 mM Tris. pH 8.0, 100 mM NaCl, 1 mMEDTA, 5 mM EGTA, and 1 mM DTT. The cells were lysed in a French Press,insoluble material removed by centrifugation, and the supernatant wasadjusted to 150 mM NaCl, 5% glycerol, 0.1% Tween 20,4 mM DTT. Thebiotinylated mCREBa was purified by addition of streptavidin-agarose(Promega) and incubation for 4 hours at 4° C. with rocking. Boundprotein was washed three times with buffer containing 50 mM Tris, pH8.0,100 mM NaCI, 1 mM EDTA, 5 mM EGTA, and 1 mM DTT. Glycerol was added to afinal concentration of 10% and the biotin-mCREBa protein bound tostreptavidin-agarose stored at −70° C.

Example 7 Determination of the DNA Binding Site of mCREBa

The binding specificity of mCREBa for DNA sequences is determined usingBinding Site Selection as described by Pollock and Treisman [NucleicAcids Res. 18:6197-6204 (1990)] which permits rapid identification ofthe DNA binding site of a protein from random oligonucleotides. Toperform Binding Site Selection of the mCREBa site, the biotin-mCREBaprotein prepared as described above is incubated with a 26 nucleotiderandom oligomer flanked by specific nucleotides that will anneal tooligonucleotide primers in a PCR reaction.

Random olionucleotide SEQ ID NO: 7

5′CAGGTCAGTTCAGCGGATCCTGTCG-(A/G/C /T)₂₆GAGGCGAATTCAGTGCAACTGCAGC-3′

The binding reaction is carried out under conditions of low ionicstrength to avoid interrupting salt-sensitive binding reactions. Bindingbuffer containing Dignam's buffer D [Digman, et al., Nucl. Acids Res.11: 1475-1489 (1983)] with protease inhibitors, 0.1% Nonidet P-40, 1mg/ml acetylated bovine serum albumin (Promega), is combined with 200 ngPoly (dIdC)-Poly (dIdC) (Pharmacia), 1 pg biotin-mCREBa, and 0.4 ngrandom oligonucleotide and incubation carried out for thirty minutes onice to permit protein/DNA complex formation. Streptavidin agarose isadded to the binding reaction and following incubation overnight at 4°C. with rocking, mCREBa/DNA complexes formed are collected bycentrifugation. Complexes are washed two times with the above bindingbuffer and DNA eluted from complex by incubation at 45° C. in buffercontaining 200 μl 5 mM EDTA, 0.5% SDS, 100 mM sodium acetate, 50 mMTris, pH 8.0. DNA is phenol extracted, mixed with 10 μg of rabbit muscleglycogen (Boehringer Mannheim) as carrier, and ethanol precipitated.Recovery of DNA is quantitated by Cerenkov counting.

The recovered DNA is amplified in a 10 pl PCR reaction including 150 μgeach of primers F and R (SEQ ID NOs: 8 and 9, respectively), 5 μCi ³²p-dCTP, 20 μM dCTP, 50μeach dATP, dGTP, dTTP, 1 mg/ml bovine serumalbumin, 1X PCR buffer (Perkin-Elmer Cetus), 1.5 mM MgCl₂, and 1 unitAmpliTaq (Perkin-Elmer Cetus).

Primer F SEQ ID NO:8 5′-CAGGTCAGTTCAGCGGATCCTGTCG-3′ Primer R SEQ IDNO:9 5′-GCTGCAGTTGCACTGAATTCGCCTC-3′

Amplification is carried out with an initial denaturation incubation at94° C. for four minutes followed by 15 cycles of 94° C. for one minute,62° C. for one minute, and 72° C. for one minutes. The product of thisPCR reaction is gel purified by after electrophoresis on a 8%nondenaturing polyacrylamide gel and used (instead of the randomoligonucleotide) for reselection of DNA binding sequences as describedabove. This may be repeated 3 or 4 times. The DNA final products aredigested with BamHI and EcoRI which flank the defined DNA binding siteand cloned into the BamHI and EcoRIsites in the polylinker region of thevector pGL2-promoter (Promega). Plasmid p GL2promoter is a vector thatexpresses firefly luciferase under control of the SV40 early promoterwith a polylinker region upstream from the promoter that permitsinsertion of potential regulatory sequences. The resulting plasmids aresubjected to DNA sequence analysis to determine a consensus nucleotidesequence of the DNA binding site for mCREBa.

DNA binding of mCREBa to the defined sequence is confirmed using a gelmobility shift assay. Briefly, purified biotin-mCREBa is incubated for15 minutes at 2 1° C. with a 0.1 pmol/μl ³²P-labelled oligonucleotidecontaining a DNA binding site identified as described above in buffercontaining 50 mM Tris, pH8, 50 mM NaCl, 50 μM DTT, 1 mM EDTA, 10%glycerol, 5 mM MgCl₂, 5 mM spermidine, 0.05% Nonidet P-40, 3 pg bovineserum albumin, and 1 μg poly(dIdC)-poly(dIdC) (Pharmacia). Afterappropriate incubation, the binding mixture is loaded onto a 4%nondenaturing polyacryla ride gel and mCREBa binding to a radiolabelledDNA sequence determined by a higher molecular weight shift of the DNA ascompared to control DNA in the absence of mCREBa protein.

As further characterization of mCREBa binding to previously identifiedDNA sequences, pGL2 constructs containing the mCREBa DNA binding site iscotransfected into 293T cells with pcDNA3-mCREBa and the ability ofexpressed mCREBa to either activate or repress transcription of thereporter construct is determined. Cells are transfected by the method ofChen and Okyama [supra], harvested after 48 hours and assayed forluciferase activity using a Luciferase Assay Kit (Promega) according tomanufacturer's suggested protocol.

Example 8 High Throughput Screen for Modulators of mCREBa DNA BindingActivity

An assay for high throughput screening of small molecule inhibitors(combinatorial libraries, natural product libraries, and/or chemicallibraries) is established based on the defined DNA binding site ofmCREBa determined above. A filter binding assay is designed in which theability of recombinant mCREBa to bind to a ³²P-labeled DNA sequence ismonitored by quantitating radioactivity bound to protein immobilized onnitrocellulose filter. The ³²P-labeled DNA is designed to besufficiently small in order that it will not bind to nitrocellulose inthe absence of previously immobilized the mCREBa protein. Putativemodulators are incubated with the immobilized protein and modulators ofDNA binding activity are identified as those which effect an increase ordecrease in the ability of the DNA to bind to the protein.

An alternative assay is established in which recombinant biotinylatedmCREBa is bound to streptavidin-coated plates (Pierce), incubated withcandidate small molecule modulators and the ³²P-labeled DNA sequenceadded. Modulators are defined as those molecules that increase ordecrease binding of mCREBa to the labeled DNA sequence.

Secondary assays involve treating cells transfected withpGL2-promoter/DNA binding site constructs with defined modulatorsfollowed by assay of activity of the product of the reporter gene.

Example 9 Identification of Modulators of mCREBa Binding to OtherProteins

Co-pending U.S. patent application Ser. No. 08/721, 684 (Attorney DocketNumber 27866/33487, filed concurrently herewith) describes in detail a“split hybrid assay” technique to identify molecules which disruptspecific protein/protein interaction. The specification of thatapplication is incorporated herein by reference.

In order to isolate cDNAs which encode proteins that interact with CKIδ,the two hybrid assay was performed using a LexA-CKIδ fusion protein asbait. The coding region of CKIδ was sub dloned into a BamHI site ofpBTM116 and transformed into a yeast strain designated CKIδb/L40 (MAT ahis3 Δ200 trp1-901 leu2-3 112 ade2 LYS::(lexAop)₄HlS3URA3::(lexAop)₈,-1cZ GAL 4). CKIδ/L40 was subjected to a large scaletransformation with a cDNA library made from mouse embryos staged atdays 9.5 and 10.5. Approximately 40 million transformants were obtained.Eighty-eight million were plated onto selective media lacking leucine,tryptophan and histidine. The ability of yeast transformants to grow inthe absence of histidine suggested that there was an interaction betweenCKIδ and some library protein.

In a second screening, interaction of the two proteins was assayed bythe ability of the interaction to activate transcription ofβ-galactosidase. Colonies that turned blue in the presence of X-gal werestreaked onto media lacking leucine, tryptophan and histidine, grown upin liquid culture and pooled for isolation of total DNA. Isolated DNAwas used to transform E. coli strain 600 which lacks the ability to growon media lacking leucine. Colonies that grew were used for plasmidpreparation and three classes of cDNA were identified. One class wasclosely related to a Drosophila transcription factor dCREBa.

When CKIδ/CREB interaction was examined in the split hybrid assay, cellswere shown to grow on media containing histidine, but in the absence ofhistidine, growth was inhibited. Addition of small amounts oftetracycline to the cell culture restored the cell's ability to grow,suggesting that the interaction between CKIδ and CREBa was very weak.

In order to identify molecules capable of disrupting binding interactionbetween mCREBa and CKIδHU, the split hybrid assay is employed in thepresence of putative inhibitors selected from, for example, chemicallibraries, natural product libraries, and combinatorial libraries.Similarly, inhibitors of interaction between mCREBa and otherinteracting kinases, as well as other proteins, are also identified inthe same techniques. Expression plasmids for other mCREBa-interactingproteins are constructed using techniques similar to those used inconstruction of a CKIHU expression plasmid. The split hybrid assay isthen performed in the presence of various putative binding inhibitorswherein inhibition is determined by the ability of the host cell to growon media lacking a nutritional requirement.

In the split hybrid assay, mCREBa, or a protein binding domain fragmentthereof, is expressed in a host cell as a fission protein in combinationwith either a DNA binding or transactivating domain of one or moretranscription factors. Examples of mCREBa binding partners include cdc2[see review in Hall and Peters, Adv. Cancer Res. 68:67-108 (1996)], CKIand CKII isoforms [see review in Ahmed, Cell Mol. Biol. Res. 40:1-11(1994)], MAP kinase [see review in Marshall, Ann. Oncol 6: Suppl.1:63-67 (1995)], and S6 kinase [see review in Chou and Blenis, Curr.Opin. Cell. BioL 7:806-814 (1995)]. A natural binding partner of mCREBais also expressed in the same host cell as a fusion protein incombination with either a DNA binding or transactivating domain of atranscription factor (whichever is not incorporated into the mCREBafusion protein). Expression of the two fusion proteins, and subsequentassociation of the binding partners permits association of the DNAbinding and transactivating domains forming an active transcriptionfactor that leads to expression of a repressor protein. The expressedrepressor protein, in turn, prevents expression of a reporter gene, thussignificantly decreasing survival of the host cell. The thus transformedhost cells can then be contacted with putative inhibitors ofmCREBa/binding partner interaction an d . actual inhibitors identifiedas those which prevent mCREBa binding to its partner protein, thuspreventing expression of the prepressor protein which cannot thereforeblock expression of the reporter gene. An inhibitor therefore indirectlyprovides for a positive signal generated by expression and detection ofthe particular reporter gene product. Sources of potential inhibitorsamenable to this type of assay can be found in chemical compoundlibraries, libraries of natural products isolated from microorganisms,animals, plants, and/or marine organisms, multiparallel syntheticcollections, and/or combinatorial, recombinatorial, peptidomimetic,peptide, polypeptide or protein libraries.

10 3190 base pairs nucleic acid single linear cDNA unknown CDS 304..18661 GGCACGAGGG ACTTTCTTGG GATGAGCGCT GCCTTTTTGG CTTCCTTTTG GATGCACAGC 60CCGATTTAAC CCCTGCACCT TCCGCCCGAT CCCAGCAGGC TTGTCCTCCC CGGGGAGTCA 120CAGATTTCCG AGGACAAGGG TCGCGTAGCC TTCGGCAGGG CTCTCCCGAG TTCCTGCTCC 180AGTGCATAAG TTCCACGCGC GCACACGCCA AGTACACGGG GAGAAGCGTC TCACCGGCCC 240GCGGCGGCTC TGCGCGGTCC CCTCCTGCCT CAGCATCCTC GGGCCTGCGC GGCGCCCACC 300GCC ATG GAG GTG CTG GAG AGC GGG GAG CAG AGC GTC CTG CAG TGG GAC 348 MetGlu Val Leu Glu Ser Gly Glu Gln Ser Val Leu Gln Trp Asp 1 5 10 15 CGCAAG CTG AGC GAG CTG TCA GAG CCC GGA GAG ACT GAG GCC CTC ATG 396 Arg LysLeu Ser Glu Leu Ser Glu Pro Gly Glu Thr Glu Ala Leu Met 20 25 30 TAC CACACG CAC TTC TCG GAG CTC CTA GAC GAG TTT TCC CAG AAC GTC 444 Tyr His ThrHis Phe Ser Glu Leu Leu Asp Glu Phe Ser Gln Asn Val 35 40 45 CTG GGT CAGCTC CTG AGT GAC CCT TTC CTC TCA GAG AAG AGC GAG TCA 492 Leu Gly Gln LeuLeu Ser Asp Pro Phe Leu Ser Glu Lys Ser Glu Ser 50 55 60 ATG GAG GTG GAGCCA TCT CCA ACA TCA CCA GCG CCT CTC ATC CAG GCT 540 Met Glu Val Glu ProSer Pro Thr Ser Pro Ala Pro Leu Ile Gln Ala 65 70 75 GAA CAC AGC TAC TCTCTG AGC GAG GAG CCC CGG ACT CAG TCA CCA TTT 588 Glu His Ser Tyr Ser LeuSer Glu Glu Pro Arg Thr Gln Ser Pro Phe 80 85 90 95 ACC CAT GCG GCT ACCAGC GAC AGC TTC AAT GAC GAG GAG GTG GAG AGT 636 Thr His Ala Ala Thr SerAsp Ser Phe Asn Asp Glu Glu Val Glu Ser 100 105 110 GAA AAA TGG TAC CTGTCT ACA GAG TTT CCT TCA GCT ACC ATC AAG AAA 684 Glu Lys Trp Tyr Leu SerThr Glu Phe Pro Ser Ala Thr Ile Lys Lys 115 120 125 GAG CCA ATC ACA GAGGAG CAG CCC CCG GGA CTT GTC CCT TCT GTC ACT 732 Glu Pro Ile Thr Glu GluGln Pro Pro Gly Leu Val Pro Ser Val Thr 130 135 140 CTG ACC ATC ACA GCCATT TCC ACT CCT TTT GAA AAA GAA GAG TCC CCT 780 Leu Thr Ile Thr Ala IleSer Thr Pro Phe Glu Lys Glu Glu Ser Pro 145 150 155 CTG GAT ATG AAT GCTGGG GGG GAC TCC TCA TGC CAG ACG CTT ATT CCT 828 Leu Asp Met Asn Ala GlyGly Asp Ser Ser Cys Gln Thr Leu Ile Pro 160 165 170 175 AAG ATT AAG CTGGAG CCC CAC GAA GTG GAT CAG TTC TTA AAC TTC TCC 876 Lys Ile Lys Leu GluPro His Glu Val Asp Gln Phe Leu Asn Phe Ser 180 185 190 CCG AAA GAA GCCTCC GTG GAT CAA CTG CAC TTA CCA CCA ACA CCA CCC 924 Pro Lys Glu Ala SerVal Asp Gln Leu His Leu Pro Pro Thr Pro Pro 195 200 205 AGT AGT CAC AGCAGT GAC TCT GAG GGC AGC TTG AGC CCC AAC CCA CGC 972 Ser Ser His Ser SerAsp Ser Glu Gly Ser Leu Ser Pro Asn Pro Arg 210 215 220 CTG CAT CCC TTCAGC CTG TCT CAG GCC CAC AGC CCT GTC AGA GCC ATG 1020 Leu His Pro Phe SerLeu Ser Gln Ala His Ser Pro Val Arg Ala Met 225 230 235 CCC CGG GGC CCCTCT GCC TTG TCC ACA TCT CCT CTC CTC ACA GCT CCA 1068 Pro Arg Gly Pro SerAla Leu Ser Thr Ser Pro Leu Leu Thr Ala Pro 240 245 250 255 CAT AAG CTGCAG GGA TCG GGC CCC CTG GTC CTG ACA GAA GAG GAG AAG 1116 His Lys Leu GlnGly Ser Gly Pro Leu Val Leu Thr Glu Glu Glu Lys 260 265 270 AGG ACC CTGGTT GCC GAG GGC TAT CCC ATT CCC ACC AAG CTG CCT CTG 1164 Arg Thr Leu ValAla Glu Gly Tyr Pro Ile Pro Thr Lys Leu Pro Leu 275 280 285 ACA AAA TCTGAG GAG AAG GCC CTG AAG AAA ATC CGG AGA AAG ATC AAG 1212 Thr Lys Ser GluGlu Lys Ala Leu Lys Lys Ile Arg Arg Lys Ile Lys 290 295 300 AAT AAG ATTTCT GCC CAA GAA AGC AGG AGA AAG AAG AAA GAA TAC ATG 1260 Asn Lys Ile SerAla Gln Glu Ser Arg Arg Lys Lys Lys Glu Tyr Met 305 310 315 GAC AGC CTGGAG AAA AAA GTG GAG TCT TGT TCA ACT GAG AAC TTG GAG 1308 Asp Ser Leu GluLys Lys Val Glu Ser Cys Ser Thr Glu Asn Leu Glu 320 325 330 335 CTT CGGAAG AAG GTG GAG GTG CTG GAG AAC ACC AAT AGG ACT CTC CTT 1356 Leu Arg LysLys Val Glu Val Leu Glu Asn Thr Asn Arg Thr Leu Leu 340 345 350 CAG CAACTT CAG AAG CTT CAG ACT TTG GTG ATG GGG AAG GTC TCT CGA 1404 Gln Gln LeuGln Lys Leu Gln Thr Leu Val Met Gly Lys Val Ser Arg 355 360 365 ACC TGCAAG TTA GCT GGC ACA CAG ACT GGC ACC TGC CTC ATG GTC GTT 1452 Thr Cys LysLeu Ala Gly Thr Gln Thr Gly Thr Cys Leu Met Val Val 370 375 380 GTG CTTTGC TTT GCT GTT GCA TTT GGA AGC TTC TTT CAA GGC TAT GGG 1500 Val Leu CysPhe Ala Val Ala Phe Gly Ser Phe Phe Gln Gly Tyr Gly 385 390 395 CCT TATCCT TCT GCC ACC AAG ATG GCT CTG CCC AGC CAG CAT CCT CTG 1548 Pro Tyr ProSer Ala Thr Lys Met Ala Leu Pro Ser Gln His Pro Leu 400 405 410 415 TCAGAG CCA TAC ACA GCC TCC GTG GTG AGA TCC AGG AAC CTG CTA ATC 1596 Ser GluPro Tyr Thr Ala Ser Val Val Arg Ser Arg Asn Leu Leu Ile 420 425 430 TATGAG GAA CAC GCT CCC CTG GAA GAG TCG TCG AGC CCA GCC TCA ACC 1644 Tyr GluGlu His Ala Pro Leu Glu Glu Ser Ser Ser Pro Ala Ser Thr 435 440 445 GGGGAG CTG GGG GGC TGG GAC AGA GGC TCC TCT CTG CTC AGG GCA TCG 1692 Gly GluLeu Gly Gly Trp Asp Arg Gly Ser Ser Leu Leu Arg Ala Ser 450 455 460 TCGGGG CTT GAG GCC CTG CCA GAG GTG GAT CTT CCC CAT TTC CTT ATC 1740 Ser GlyLeu Glu Ala Leu Pro Glu Val Asp Leu Pro His Phe Leu Ile 465 470 475 TCCAAT GAG ACG AGC TTG GAG AAG TCA GTA CTG TTG GAG CTT CAG CAG 1788 Ser AsnGlu Thr Ser Leu Glu Lys Ser Val Leu Leu Glu Leu Gln Gln 480 485 490 495CAC CTG GTC AGC AGC AAA CTG GAA GGG AAC GAA ACA CTC AAG GTT GTA 1836 HisLeu Val Ser Ser Lys Leu Glu Gly Asn Glu Thr Leu Lys Val Val 500 505 510GAG CTG GAG AGG AGA GTG AAC GCC ACC TTC TGAGGAGAGC TCCACCCTCC 1886 GluLeu Glu Arg Arg Val Asn Ala Thr Phe 515 520 TCTTCTCCTA ACTCCATCTGATCGTCCTTT CAGTTTCCCC TTCACCACTG GATCTCGA 1946 AGGAGATGGC TAGTGTTACGGCTCGAGACA GGAGGCCAGC CCAGGGGGTT CTGCTTAT 2006 GTCCCCGTGG CTCTCCACAAAAGGGAGCTA GCACCTCTCC ATCCCTTTCT CTTACTGC 2066 TTGGAAATTA TTTTAGGGCTGAGATAGGGG TGGAACGAGC AGGCTTGTTT CCACCAAT 2126 TGCCAAGAAG ACACTGCCTGATTCTTCCCC GGGAGGAGTG ACTCCTCTGA AGAAGACA 2186 ACTCATGTTC AGTTGAGACCCCAGACTCTA GCCACACACA TGCCACAGAC ATGCCAGG 2246 GTGGCAAAGC ACTGACTCCTGAGCTCCCTT CCTCACTAGG ACTCCAGTGT GACCCTGC 2306 TGAGAGGACC AAAGCGTCATTGCAGTCTTC TCTCCACCCT GTACCCCGGA GTCCTGAT 2366 GATGTCTGCA GAGGCAGATGGGGCTCCCAC CATATTTTCA GGCCGCAAGT GCAATTCC 2426 AAGGCATCAG GCTCTTCTCTCCCAGGCTCT CCTGCCCACT GTGTTGTTTG TAGGACAC 2486 CCACACCCAC TCATACACAGCCTGCATCTC CACAGGACAA TAGCTCTGTC TCCCTGGC 2546 CCCCTCCCCA TTTGTAAATAGTATTTATTA GCTTGCTCAA GCTCCCAGCT GGCCATAG 2606 AAAAGATTTC CCCTTTCAACCAGCAAAGTC TTCTGTTGGC CTTTGGAACA GGAGAGTC 2666 CGGAATCTAG GACCCTAGTCTTTGTACTTG ATGCCTTGTT TCCCCCCTTT TCTTCTTT 2726 AATTGGGGAC CTATAACATCATCGCTGTTG CGGAATCCAC TTAGGCATGT GTCCCCTG 2786 GGATGAATAC ATGGGAATGGTGGATACTGT CTTCTGACTC AGGCTCTAGG CTCCATGG 2846 TCCTCTCTCT GGTCCTGCCACACAGAAGGA AAGCCCTGTC CAGGATAATG AGCGTTGC 2906 ACACCCTTGC TAGCTTGTCCTGCCTACCTG CTTACCCCAC TCCCTCACCT TCCTCCTT 2966 CTTCTGCCCT CCATCCACCTGCCTTAACTA ATTGGGGCTG GAGTTGGTCA TTTTTTGT 3026 ACCCACAGTG GTACCTTTTACAGTCAGGTT TGGATACTTT GCAGCTCATC CAAAGAGA 3086 TAACTAAACC CTAAACTCTTTTTTTGTTGT TGTTGTTGTT GTTTTTTTTT TTTATGAT 3146 AAAAGTAAAA ATTGTAGTTTAAAAAAAAAA AAAAAAAACT CGAG 3190 521 amino acids amino acid linearprotein unknown 2 Met Glu Val Leu Glu Ser Gly Glu Gln Ser Val Leu GlnTrp Asp Arg 1 5 10 15 Lys Leu Ser Glu Leu Ser Glu Pro Gly Glu Thr GluAla Leu Met Tyr 20 25 30 His Thr His Phe Ser Glu Leu Leu Asp Glu Phe SerGln Asn Val Leu 35 40 45 Gly Gln Leu Leu Ser Asp Pro Phe Leu Ser Glu LysSer Glu Ser Met 50 55 60 Glu Val Glu Pro Ser Pro Thr Ser Pro Ala Pro LeuIle Gln Ala Glu 65 70 75 80 His Ser Tyr Ser Leu Ser Glu Glu Pro Arg ThrGln Ser Pro Phe Thr 85 90 95 His Ala Ala Thr Ser Asp Ser Phe Asn Asp GluGlu Val Glu Ser Glu 100 105 110 Lys Trp Tyr Leu Ser Thr Glu Phe Pro SerAla Thr Ile Lys Lys Glu 115 120 125 Pro Ile Thr Glu Glu Gln Pro Pro GlyLeu Val Pro Ser Val Thr Leu 130 135 140 Thr Ile Thr Ala Ile Ser Thr ProPhe Glu Lys Glu Glu Ser Pro Leu 145 150 155 160 Asp Met Asn Ala Gly GlyAsp Ser Ser Cys Gln Thr Leu Ile Pro Lys 165 170 175 Ile Lys Leu Glu ProHis Glu Val Asp Gln Phe Leu Asn Phe Ser Pro 180 185 190 Lys Glu Ala SerVal Asp Gln Leu His Leu Pro Pro Thr Pro Pro Ser 195 200 205 Ser His SerSer Asp Ser Glu Gly Ser Leu Ser Pro Asn Pro Arg Leu 210 215 220 His ProPhe Ser Leu Ser Gln Ala His Ser Pro Val Arg Ala Met Pro 225 230 235 240Arg Gly Pro Ser Ala Leu Ser Thr Ser Pro Leu Leu Thr Ala Pro His 245 250255 Lys Leu Gln Gly Ser Gly Pro Leu Val Leu Thr Glu Glu Glu Lys Arg 260265 270 Thr Leu Val Ala Glu Gly Tyr Pro Ile Pro Thr Lys Leu Pro Leu Thr275 280 285 Lys Ser Glu Glu Lys Ala Leu Lys Lys Ile Arg Arg Lys Ile LysAsn 290 295 300 Lys Ile Ser Ala Gln Glu Ser Arg Arg Lys Lys Lys Glu TyrMet Asp 305 310 315 320 Ser Leu Glu Lys Lys Val Glu Ser Cys Ser Thr GluAsn Leu Glu Leu 325 330 335 Arg Lys Lys Val Glu Val Leu Glu Asn Thr AsnArg Thr Leu Leu Gln 340 345 350 Gln Leu Gln Lys Leu Gln Thr Leu Val MetGly Lys Val Ser Arg Thr 355 360 365 Cys Lys Leu Ala Gly Thr Gln Thr GlyThr Cys Leu Met Val Val Val 370 375 380 Leu Cys Phe Ala Val Ala Phe GlySer Phe Phe Gln Gly Tyr Gly Pro 385 390 395 400 Tyr Pro Ser Ala Thr LysMet Ala Leu Pro Ser Gln His Pro Leu Ser 405 410 415 Glu Pro Tyr Thr AlaSer Val Val Arg Ser Arg Asn Leu Leu Ile Tyr 420 425 430 Glu Glu His AlaPro Leu Glu Glu Ser Ser Ser Pro Ala Ser Thr Gly 435 440 445 Glu Leu GlyGly Trp Asp Arg Gly Ser Ser Leu Leu Arg Ala Ser Ser 450 455 460 Gly LeuGlu Ala Leu Pro Glu Val Asp Leu Pro His Phe Leu Ile Ser 465 470 475 480Asn Glu Thr Ser Leu Glu Lys Ser Val Leu Leu Glu Leu Gln Gln His 485 490495 Leu Val Ser Ser Lys Leu Glu Gly Asn Glu Thr Leu Lys Val Val Glu 500505 510 Leu Glu Arg Arg Val Asn Ala Thr Phe 515 520 29 base pairsnucleic acid single linear DNA unknown 3 CGCGGATCCT AATGGAGCTG AGAGTCGGG29 29 base pairs nucleic acid single linear DNA unknown 4 CGCGGATCCGCTCATCGGTG CACGACAGA 29 29 base pairs nucleic acid single linear DNAunknown 5 CGGGATCCTC ACAGCTCCAC ATAAGCTGC 29 28 base pairs nucleic acidsingle linear DNA unknown 6 GGAATTCGCT CAAGGAGAGT CCTATTGG 28 154 basepairs nucleic acid single linear DNA unknown 7 CAGGTCAGTT CAGCGGATCCTGTCGNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 60 NNNNNNNNNN NNNNNNNNNNNNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 120 NNNNNNNNNG AGGCGAATTCAGTGCAACTG CAGC 154 25 base pairs nucleic acid single linear DNA unknown8 CAGGTCAGTT CAGCGGATCC TGTCG 25 25 base pairs nucleic acid singlelinear DNA unknown 9 GCTGCAGTTG CACTGAATTC GCCTC 25 38 base pairsnucleic acid single linear DNA unknown 10 GAATCGGGCC GCCGAGATCTCATATGGAGC TGAGAGTC 38

What is claimed is:
 1. A method to identify modulators of mCREBa bindingto DNA comprising the steps of: a) incubating mCREBa with a DNA sequenceknown to specifically bind mCREBa; b) determining the degree of bindingbetween mCREBa and the DNA sequence; c) repeating step (a) in thepresence of a putative modulator of mCREBa binding to the DNA sequences;and d) comparing binding of mCREBa with the DNA sequence in the presenceof the putative modulator in step (c) to the degree of bindingdetermined in step (b), and identifying the putative modulator as anenhancer when increased binding is detected in the presence of theputative modulator and identifying the putative modulator as a repressorwhen decreased binding in the presence of the putative modulator isdetected.
 2. A method to identify an inhibitor of binding between mCREBaor binding fragment thereof and a mCREBa-binding protein or bindingfragment thereof comprising the steps of: a) producing host cellstransformed or transfected with DNA comprising: a repressor geneencoding a repressor protein, said repressor gene under transcriptionalcontrol of a promoter; a selectable marker gene encoding a selectablemarker protein; said selectable marker gene under transcriptionalcontrol of an operator; said operator regulated by interaction with saidrepressor protein; a first recombinant fusion protein gene encodingmCREBa or a binding fragment thereof in frame with either a DNA bindingdomain of a transcriptional activating protein or a transactivatingdomain of a transcriptional activating protein; and a second recombinantfusion protein gene encoding a mCREBa-binding protein or bindingfragment thereof in frame with either a DNA binding domain of atranscriptional activating protein or a transactivating domain of atranscriptional activating protein, whichever domain is not encoded bythe first fusion protein gene, said second binding protein or bindingfragment thereof capable of interacting with said first binding proteinor binding fragment thereof such that interaction of said second bindingprotein or binding fragment thereof and said first binding protein orbinding fragment thereof brings into proximity a DNA binding domain anda transactivating domain forming a functional transcriptional activatingprotein; said functional transcriptional activating protein acting onsaid promoter to increase expression of said repressor gene. b) growingthe host cells in the absence of a test compound and under conditionswhich permit expression of said mCREBa or binding fragment thereof andsaid mCREBa-binding protein or binding fragment thereof such that saidmCREBa or fragment thereof and said mCREBa binding protein or bindingfragment thereof interact bringing into proximity said DNA bindingdomain and said transactivating domain forming said functionaltranscriptional activating protein; said transcriptional activatingprotein acting on said promoter to increase expression of said repressorprotein; said repressor protein interacting with said operator such thatsaid selectable marker protein is not expressed; c) confirming lack ofexpression of said selectable marker protein in said host cell; d)growing said host cells in the presence of a test compound; e)determining expression of said selectable marker protein in the presenceof said test compound, and f ) comparing expression of said selectablemarker protein in the presence and absence of said test compound whereinincreased expression of said selectable marker protein is indicativethat the test compound is an inhibitor of binding between said firstbinding protein or binding fragment thereof and said second bindingprotein or binding fragment thereof.
 3. The method of claim 2 whereinsaid DNA binding domain and said transactivating domain are derived froma common transcriptional activating protein.
 4. The method of claim 2wherein one or more of the repressor gene, the selectable marker gene,the first recombinant fusion protein gene, and the second recombinantfusion protein gene are encoded on distinct DNA expression constructs.5. The method of claim 2 wherein said selectable marker protein is anenzyme in a pathway for synthesis of a nutritional requirement for saidhost cell such that expression of said selectable marker protein isrequired for growth of said host cell on media lacking said nutritionalrequirement.
 6. The method of claim 2 wherein said host cell is a yeastcell or a mammalian.
 7. The method of claim 3 wherein said selectablemarker gene encodes HIS3.
 8. The method of claim 3 wherein saidrepressor protein gene encodes a tetracycline resistance protein.
 9. Themethod of claim 3 wherein said operator is a tet operator.
 10. Themethod of claim 3 wherein said promoter is selected from the groupconsisting of the LexA promoter, the alcohol dehydrogenase promoter, theGal4 promoter.
 11. The method of claim 3 wherein said DNA binding domainderived from a protein selected from the group consisting of LexA andGal4.
 12. The method of claim 3 wherein said transactivating domain isderived from a protein selected from the group consisting of VP16 andGal4.
 13. The method of claim 3 wherein the mCREBa-binding protein isselected from the group consisting of CKI, CKII, cdc2, MAP kinase, andS6 kinase.